4-aryl-3-hydroxyquinolin-2-one derivatives as ion channel modulators

ABSTRACT

There is provided novel substituted 4-aryl-3-hydroxyquinolin-2-one derivatives of the formula ##STR1## wherein R is hydrogen or methyl; 
     R 1 , R 2 , R 3  and R 4  each are independently hydrogen, bromo, chloro or trifluoromethyl, and when R 1 , R 3  and R 4  are hydrogen, R 2  is nitro; 
     R 5  is hydrogen or methyl; and 
     R 6  is bromo or chloro; 
     or a nontoxic pharmaceutically acceptable salt thereof, which are openers of the large-conductance calcium-activated potassium channels and are useful in the treatment of disorders which are responsive to the opening of the potassium channels.

CROSS-REFERENCE TO RELATED APPLICATIONS

This nonprovisional application claims the benefit of copendingprovisional application, U.S. Ser. No. 60/031,105 filed Nov. 26, 1996.

FIELD OF THE INVENTION

The present invention is directed to novel4-aryl-3-hydroxyquinolin-2-one derivatives which are modulators of thelarge-conductance calcium-activated potassium (BK) channels and,therefore, useful in the protection of neuronal cells and diseasesarising from dysfunction of cellular membrane polarization andconductance.

BACKGROUND OF THE INVENTION

Potassium channels play a key role in regulation of cell membranepotential and modulation of cell excitability. Potassium channels arelargely regulated by voltage, cell metabolism, calcium and receptormediated processes. Cook, N. S., Trends in Pharmacol. Sciences (1988),9, 21; and Quast, U., et al, Trends in Pharmacol. Sciences (1989), 10,431!. Calcium-activated potassium (K_(Ca)) channels are a diverse groupof ion channels that share a dependence on intracellular calcium ionsfor activity. The activity of K_(Ca) channels is regulated byintracellular Ca²⁺ !, membrane potential and phosphorylation. On thebasis of their single-channel conductances in symmetrical K⁺ solutions,K_(Ca) channels are divided into three subclasses: large conductance(BK)>150 pS; intermediate conductance 50-150 pS; small conductance<50pS. Large-conductance calcium-activated potassium (Maxi-K or BK)channels are present in many excitable cells including neurons, cardiaccells and various types of smooth muscle cells. Singer, J. et al.,Pflugers Archiv. (1987) 408, 98; Baro, I., et al., Pflugers Archiv.(1989) 414 (Suppl. 1), S168; and Ahmed, F. et al., Br. J. Pharmacol.(1984) 83, 227!.

Potassium ions play a dominant role in controlling the resting membranepotential in most excitable cells and maintain the transmembrane voltagenear the K⁺ equilibrium potential (E_(k)) of about -90 mV. It has beenshown that opening of potassium channels shift the cell membranepotential towards the equilibrium potassium membrane potential (E_(k)),resulting in hyperpolarization of the cell. Cook, N. S., Trends inPharmacol. Sciences (1988), 9, 21!. Hyperpolarized cells show a reducedresponse to potentially damaging depolarizing stimuli. BK channels whichare regulated by both voltage and intracellular Ca²⁺ act to limitdepolarization and calcium entry and may be particularly effective inblocking damaging stimuli. Therefore cell hyperpolarization via openingof BK channels may result in protection of neuronal cells.

A range of synthetic and naturally occurring compounds with BK openingactivity have been reported. The avena pyrone extracted from avenasativa-common oats has been identified as a BK channel opener usinglipid bi-layer technique International Patent application WO 93/08800,published May 13, 1993!. 6-Bromo-8-(methylamino) imidazo1,2-a!pyrazine-2-carbonitrile (SCA-40) has been described as a BKchannel opener with very limited electrophysiological experimentsLaurent, F. et al., Br. J. Pharmacol. (1993) 108, 622-626!. Theflavanoid, Phloretin has been found to increase the open probability ofCa²⁺ -activated potassium channels in myelinated nerve fibers of Xenopuslaevis using outside-out patches Koh, D-S., et al., Neuroscience Lett.(1994) 165, 167-170!.

In European patent application EP-477,819 published Jan. 4, 1992 andcorresponding U.S. Pat. No. 5,200,422, issued Apr. 6, 1993 to Olesen, etal., a number of benzimidazole derivatives were disclosed as openers ofBK channels by using single-channel and whole-cell patch-clampexperiments in aortic smooth muscle cells. Further work was reported byOlesen, et al in European J. Pharmacol., 251, 53-59 (1994).

A number of substituted oxindoles have been disclosed as openers of BKchannels by P. Hewawasam, et al, in U.S. Pat. No. 5,565,483, issued Oct.15, 1996.

A. Walser, et al, J. Org. Chem., 38, 449-456 (1973) disclose a limitednumber of 3-hydroxyquinolinones as by-products formed during the openingof the epoxide intermediate.

Y. S. Mohammed, et al, Pharmazie, 40, 312-314 (1985) discloses a seriesof 4-substituted-3-hydroxy-2-quinolones as analogues of the naturalproduct viridicatin. The Merck Index, 11th Edition, 1575 (1989) brieflysummarizes the references to the antibiotic substance, viridicatin.

M. S. Masoud, et al, in Spectroscopy Letters, 21 (6), 369-383 (1988)describe the spectral properties of several 2-quinolones as liquids andin Synth. React. Inorg. Met.-Org. Chem.,17, (8 & 9), 881-899 (1987)describe the equilibria and stability of the 2-quinolones in metalliccomplexes.

It is the object of the present invention to provide novel compoundsthat will modulate potassium channels, in particular, large conductancecalcium-activated potassium (BK) channels which will be useful inreducing neuronal damage.

SUMMARY OF THE INVENTION

The present invention provides novel 4-aryl-3-hydroxyquinolin-2-onederivatives having the general formula ##STR2## wherein R, R¹, R², R³,R⁴, R⁵ and R⁶ are as defined below, or a non-toxic pharmaceuticallyacceptable salt thereof which are openers of the large conductancecalcium-activated K⁺ channels also known as Maxi-K or BK channels. Thepresent invention also provides pharmaceutical compositions comprisingsaid quinolin-2-one derivatives and to the method of treatment ofdisorders sensitive to potassium channel opening activity such asischemia, convulsions, asthma, irritable bowel syndrome, migraine,traumatic brain injury, and urinary incontinence.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel 4-aryl-3-hydroxyquinolin-2-onederivatives which are potent openers of the high conductance,calcium-activated K⁺ -channels (BK channel) and which have the formula##STR3## wherein R is hydrogen or methyl;

R¹, R², R³ and R⁴ each are independently hydrogen, bromo, chloro ortrifluoromethyl, and when R¹, R³ and R⁴ are hydrogen, R² is nitro;

R⁵ is hydrogen or methyl; and

R⁶ is bromo or chloro; or a nontoxic pharmaceutically acceptable saltthereof.

The present invention also provides a method for the treatment oralleviation of disorders associated with BK channels, especiallyischemia, convulsions, asthma, irritable bowel syndrome, migraine,traumatic brain injury, and urinary incontinence, which comprisesadministering together with a conventional adjuvant, carrier or diluenta therapeutically effective amount of a compound of formula I or anontoxic pharmaceutically acceptable salt thereof.

The term "nontoxic pharmaceutically acceptable salt" as used herein andin the claims is intended to include nontoxic base addition salts withinorganic bases. Suitable inorganic bases such as alkali and alkalineearth metal bases include metallic cations such as sodium, potassium,magnesium, calcium and the like.

Certain of the compounds of the present invention can exist inunsolvated forms as well as solvated forms including hydrated forms suchas monohydrate, dihydrate, hemihydrate, trihydrate, tetrahydrate and thelike. The products may be true solvates, while in other cases, theproducts may merely retain adventitious solvent or be a mixture ofsolvate plus some adventitious solvent. It should be appreciated bythose skilled in the art that solvated forms are equivalent tounsolvated forms and are intended to be encompassed within the scope ofthe present invention.

In the method of the present invention, the term "therapeuticallyeffective amount" means the total amount of each active component of themethod that is sufficient to show a meaningful patient benefit, i.e.,healing of acute conditions characterized by openers of largeconductance calcium-activated K⁺ channels or increase in the rate ofhealing of such conditions. When applied to an individual activeingredient, administered alone, the term refers to that ingredientalone. When applied to a combination, the term refers to combinedamounts of the active ingredients that result in the therapeutic effect,whether administered in combination, serially or simultaneously. Theterms "treat, treating, treatment" as used herein and in the claimsmeans preventing or ameliorating diseases, tissue damage and/or symptomsassociated with dysfunction of cellular membrane polarization andconductance.

The compounds of Formula I may be prepared by various procedures such asthose illustrated herein in the examples, in the reaction schemes andvariations thereof which would be evident to those skilled in the art.The various quinolin-2-one derivatives of Formula I may advantageouslybe prepared from isatin intermediates which are generally well-known anda general method of preparation is illustrated in Reaction Scheme 1.

In the process for the preparation of isatin intermediates of theFormula III, a number of commonly known and well-established proceduresmay be employed such as those described by Sandmeyer, T., Helv. Chim.Acta, 2, 234 (1919); Stolle, R., J. Prakt. Chem., 105, 137 (1922); andGassman, P., et al., J. Org. Chem., 42, 1344 (1977). However, a morepreferred method for the preparation of isatins of Formula III startingfrom the appropriately substituted anilines of Formula V is generallydescribed by Hewawasam, P., et al., Tetrahedron Lett., 35, 7303 (1994)and is illustrated in Reaction Scheme 1. This method appears to beinsensitive to the electronic nature of substituents bound to thearomatic ring and is characterized by predictable regiochemical control.##STR4##

It will be appreciated by those skilled in the art that when the aminogroup of an aniline compound of Formula V is suitably protected such aswith N-pivaloyl and N-(tert-butoxycarbonyl) protecting groups, it candirect metalation to the ortho position. Once the dianions are formed,the reaction with about 1.2 equivalents of diethyl oxalate at lowtemperatures such as -78° C. may be used to introduce an α-ketoestermoiety ortho to the protected amino group of the aniline derivative toproduce the compound of Formula VI. Removal of the protecting groupfollowed by spontaneous cyclization will advantageously produce theisatin of Formula III. To elaborate further on the process of ReactionScheme 1, the dianions of N-pivaloylanilines or N-(tert-butoxycarbonyl)anilines are advantageously generated using about 2.2 to 2.4 fold excessof a variety of butyllithium reagents, such as n-butyl-, s-butyl- andt-butyl-lithium reagents in THF at about 0° to -40° C. for 2 to 7 hours.

In a typical procedure, neat dry diethyl oxalate (1.2 equivalents) wasadded to a solution of the dianion stirred at -78° C. under nitrogen.After being stirred for 30-45 minutes, the reaction was quenched with 1NHCl and diluted with diethyl ether to afford the compound of Formula VI.Although the intermediate α-ketoesters of Formula VI may be purified forpurposes of characterization, this step is not necessary and the crudeproduct can be advantageously deprotected to afford the isatins inexcellent overall yield. Deprotection of the N-(tert-butoxycarbonyl) orpivaloyl moieties may be carried out using 3N HCl/THF or 12N HCl/DME,respectively, at reflux temperature. Upon evaporation of the volatilesolvents, the isatins generally precipitated from the aqueous residueand are isolated by filtration.

Isatins of Formula III, prepared as described in the above ReactionScheme 1 or by well-known literature procedures, were converted to the4-aryl-3-hydroxyquinolin-2-ones of Formulas Ia and Ib as shown inReaction Scheme 2.

In the process for the preparation of compounds of Formula Ia, thehydrazone of Formula IV is condensed with the appropriate isatin ofFormula III to produce a mixture of quinolinone regioisomers of FormulaIa and II. The hydrozones of Formula IV are advantageously prepared fromthe corresponding readily available substituted benzaldehydes. Thecondensation reaction is conducted in a C₁₋₄ alcohol solvent such asmethanol, ethanol and 2-propanol in the presence of base derived from anearth metal salt of lower alkynols such as sodium methoxide. Thereaction is advantageously conducted above room temperature andpreferably at about 65°-100° C. for about 3 to 12 hours. The resultingmixture of isomers of Formula Ia and II are suspended and heated inethyl acetate and filtered. Usually the less soluble and in mostinstances the undesirable quinolinone regioisomer of Formula II isremoved by filtration. The separation and the successful removal of theundesired isomer was ascertained by ¹ H NMR. In most cases, theseparation and removal of the undesired isomer was complete. However, ifthe separation was not complete, it is desirable to re-suspend the solidmixture in ethyl acetate to remove insoluables. This process can berepeated several times, if necessary, until a single isomer is obtained.

Alternatively, the preparation of quinolinones of the Formula Ia may becarried out from the corresponding isatin of Formula III wherein R^(a)is --CH₂ OCH₃ or CH₃ and the hydrazone of Formula IV. It has been foundthat the use of a methoxymethyl group as a protecting (blocking) groupfor R^(a) in the condensation reaction of the process illustrated inReaction Scheme 2 will advantageously produce a much higher amount ofthe desired regioisomer of Formula Ia.

Demethylation of the methyl ether moiety of the compound of Formula Iawith BBr₃ in CH₂ Cl₂ under carefully controlled conditions from -78° to0° C. afforded the desired phenols of Formula Ib. The reaction shouldpreferrably not be warmed above 0° C. After completion of thedemethylation, quenching of the reaction afforded the4-aryl-3-hydroxyquinolin-2-ones of Formula Ib. ##STR5##

In addition to the differences observed in the proton NMR spectrum forthe regioisomers of Formula Ia and II, the absolute structure of thedesired regioisomeric compound of Formula Ia and Ib was verified andconfirmed by single crystal x-ray analysis. In general, the experimental¹ H NMR spectra in DMSO-d₆ of the compound of formula Ia and Ibexhibited certain characteristic chemical shift peaks in the protonspectrum which distinguished these products from the undesiredregioisomer of formula II. The chemical shift for the 3-OH peak wasobserved at about 9.5-9.8 ppm and the NH peak was observed at about12.2-12.6 ppm while the regioisomer of formula II generally exhibitedchemical shifts for the 4-OH peak at about 10-10.5 ppm and the NH peakat about 11.5-11.8 ppm.

In a preferred embodiment of the invention the compounds of Formula Ihave the formula ##STR6## wherein R is hydrogen, R¹, R², R³ and R⁴ eachare independently hydrogen, bromo, chloro, or trifluoromethyl, and whenR¹, R³ and R⁴ are hydrogen, R² is nitro; or a nontoxic pharmaceuticallyacceptable salt thereof.

In another aspect, this invention provides a method for the treatment ofor protection from disorders which are mediated by opening of the largeconductance calcium-activated K⁺ channels (BK channels) in a mammal inneed thereof, which comprises administering to said mammal atherapeutically effective amount of a compound of Formula I or anontoxic pharmaceutically acceptable salt thereof. Preferably, thecompounds of Formula I are useful in the treatment of ischemia,convulsions, asthma, irritable bowel syndrome, migraine, traumatic braininjury, and urinary incontinence and other disorders sensitive to BKchannel activating activity.

In still another aspect, this invention provides pharmaceuticalcompositions comprising at least one compound of Formula I incombination with a pharmaceutical adjuvant, carrier or diluent.

Biological Activity

Potassium (K⁺) channels are structurally and functionally diversefamilies of K⁺ -selective channel proteins which are ubiquitous incells, indicating their central importance in regulating a number of keycell functions Rudy, B., Neuroscience, 25: 729-749 (1988)!. While widelydistributed as a class, K⁺ channels are differentially distributed asindividual members of this class or as families. Gehlert, D. R., et al.,Neuroscience, 52: 191-205 (1993)!. In general, activation of K⁺ channelsin cells, and particularly in excitable cells such as neurons and musclecells, leads to hyperpolarization of the cell membrane, or in the caseof depolarized cells, to repolarization. In addition to acting as anendogenous membrane voltage clamp, K⁺ channels can respond to importantcellular events such as changes in the intracellular concentration ofATP or the intracellular concentration of calcium (Ca²⁺). The centralrole of K⁺ channels in regulating numerous cell functions makes themparticularly important targets for therapeutic development. Cook, N. S.,Potassium channels: Structure, classification, function and therapeuticpotential. Ellis Horwood, Chinchester (1990)!. One class of K+ channels,the large-conductance Ca²⁺ -activated K⁺ channels (Maxi-K or BKchannels), is regulated by transmembrane voltage, intracellular Ca²⁺,and a variety of other factors such as the phosphorylation state of thechannel protein. Latorre, R., et al., Ann. Rev. Pysiol., 51: 385-399(1989)!. The large, single channel-conductance (generally>150 pS) andhigh degree of specificity for K⁺ of BK channels indicates that smallnumbers of channels could profoundly affect membrane conductance andcell excitability. Additionally, the increase in open probability withincreasing intracellular Ca²⁺ indicates involvement of BK channels inthe modulation of Ca²⁺ -dependent phenomena such as secretion andmuscular contraction. Asano, M., et al., J. Pharmacol. Exp. Ther., 267:1277-1285 (1993)!.

Openers of BK channels exert their cellular effects by increasing theopen probability of these channels McKay, M. C., et al., J.Neurophysiol., 71: 1873-1882 (1994); and Olesen, S.-P., Exp. Opin.Invest. Drugs, 3: 1181-1188 (1994)!. This increase in the opening ofindividual BK channels collectively results in the hyperpolarization ofcell membranes, particularly in depolarized cells, produced bysignificant increases in whole-cell BK-mediated conductance.

The ability of compounds described in the present invention to open BKchannels and increase whole-cell outward (K⁺) BK-mediated currents wasassessed under voltage-clamp conditions by determining their ability toincrease cloned mammalian (mSlo or hSlo) BK-mediated outward currentheterologously expressed in Xenopus oocytes Butler, A., et al., Science,261: 221-224 (1993); and Dworetzky, S. I., et al., Mol. Brain Res., 27:189-193 (1994)!. The two BK constructs employed represent nearlystructurally identical homologous proteins, and have proven to bepharmacologically identical in our tests. To isolate BK current fromnative (background, non-BK) current, the specific and potent BKchannel-blocking toxin iberiotoxin (IBTX) Galvez, A., et al., J. Biol.Chem., 265: 11083-11090 (1990)! was employed at a supramaximalconcentration (50 nM). The relative contribution of BK channels currentto total outward current was determined by subtraction of the currentremaining in the presence of IBTX (non-BK current) from the currentprofiles obtained in all other experimental conditions (control, drug,and wash). It was determined that at the tested concentration thecompounds profiled did not effect non-BK native currents in the oocytes.All compounds were tested in at least 5 oocytes and are reported at thesingle concentration of 20 μM; the effect of the selected compounds ofFormula I on BK current was expressed as the percent of controlIBTX-sensitive current and is listed in Table I. Recordings wereaccomplished using standard two-electrode voltage clamp techniquesStuhmer, W., et al., Methods in Enzymology, Vol. 207: 319-339 (1992)!;voltage-clamp protocols consisted of 500-750 ms duration stepdepolarization from a holding potential of -60 mV to +140 mV in 20 mVsteps. The experimental media (modified Barth's solution) consisted of(in mM): NaCl (88), NaHCO3 (2.4), KCl (1.0), HEPES (10), MgSO4 (0.82),Ca(NO3)2 (0.33), CaCl2 (0.41); pH 7.5.

                  TABLE 1    ______________________________________    Effect of Selected Compounds on BK Channels           Example                  BK           No.    Current*    ______________________________________           2      ++           3      +           4      ++           5      +           6      +           7      +           8      +           9      ++           10     ++           11     ++           12     ++           13     +           14     +           15     ++           16     ++           17     ++           18     ++           19     ++           20     ++           21     +    ______________________________________     *at 20 μM expressed as percent of controls     + = 100-150%     ++ = >150%

The results of the above biological test demonstrates that the compoundsof the instant invention are potent openers of the large-conductancecalcium-activated K⁺ channels (Maxi-K or BK channels). Thus, thecompounds of the present invention are useful for the treatment of humandisorders arising from dysfunction of cellular membrane polarization andconductance and, preferably, are indicated for the treatment ofischemia, convulsions, asthma, irritable bowel syndrome, migraine,traumatic brain injury, and urinary incontinence and other disorderssensitive to BK channel activating activity.

In another embodiment, this invention includes pharmaceuticalcompositions comprising at least one compound of Formula I incombination with a pharmaceutical adjuvant, carrier or diluent.

In still another embodiment, this invention relates to a method oftreatment or prevention of disorders responsive to opening of potassiumchannels in a mammal in need thereof, which comprises administering tosaid mammal a therapeutically effective amount of a compound of FormulaI or a nontoxic pharmaceutically acceptable salt thereof.

Antibiotic Activity

The in vitro antibacterial activity of a representative number ofcompounds from the present invention were determined by the two-foldagar dilution method. The activity against the following test organismswere evaluated.

    ______________________________________    Staphylococcus aureus/Pen. -                            A9537    Staphylococcus aureus/Pen. +                            A9606    Staphylococcus epidermidis                            A24548    Micrococcus luteus      A9852    Micrococcus luteus      A21349    Bacillus subtilis       A9506A    ______________________________________

The results using representative compounds of Examples 2, 3, 4, 6, 8, 9,10, 11 and 12 exhibited fairly good inhibitory activity with MIC valuesin the range of 0.5 to 32 μg/ml and mostly in the 1 to 2 μg/ml level ofactivity against the gram-positive test organisms.

The novel quinolinones of general Formula I or the pharmaceuticallyacceptable salts thereof, are active against various gram-positivebacteria and they may be used, for example, as animal feed additives forpromotion of growth, as preservatives in food, as bactericides inindustrial applications, for example in waterbased paint and in thewhite water of paper mills to inhibit the growth of harmful bacteria andas disinfectants for destroying or inhibiting the growth of harmfulbacteria on medical and dental equipment. They are also useful, however,in the treatment of infectious disease in animals caused bygram-positive bacteria.

In respect to pharmaceutical compositions containing the antibioticherein, carrier and other ingredients should be such as not to diminishthe therapeutic effects of the antibiotic. Suitable dosage forms willcomprise an amount effective to inhibit the growth of the bacteriacausing the condition of infection and will depend on the age and weightof the mammalian species being treated, the route of administration, andthe type and severity of the infectious condition being treated andother factors readily evaluated by the physician or veterinarian inattendance.

For therapeutic use, the pharmacologically active compounds of Formula Iwill normally be administered as a pharmaceutical composition comprisingas the (or an) essential active ingredient at least one such compound inassociation with a solid or liquid pharmaceutically acceptable carrierand, optionally, with pharmaceutically acceptable adjutants andexcipients employing standard and conventional techniques.

The pharmaceutical compositions include suitable dosage forms for oral,parenteral (including subcutaneous, intramuscular, intradermal andintravenous) bronchial or nasal administration. Thus, if a solid carrieris used, the preparation may be tableted, placed in a hard gelatincapsule in powder or pellet form, or in the form of a troche or lozenge.The solid carrier may contain conventional excipients such as bindingagents, fillers, tableting lubricants, disintegrants, wetting agents andthe like. The tablet may, if desired, be film coated by conventionaltechniques. If a liquid carrier is employed, the preparation may be inthe form of a syrup, emulsion, soft gelatin capsule, sterile vehicle forinjection, an aqueous or non-aqueous liquid suspension, or may be a dryproduct for reconstitution with water or other suitable vehicle beforeuse. Liquid preparations may contain conventional additives such assuspending agents, emulsifying agents, wetting agents, non-aqueousvehicle (including edible oils), preservatives, as well as flavoringand/or coloring agents. For parenteral administration, a vehiclenormally will comprise sterile water, at least in large part, althoughsaline solutions, glucose solutions and like may be utilized. Injectablesuspensions also may be used, in which case conventional suspendingagents may be employed. Conventional preservatives, buffering agents andthe like also may be added to the parenteral dosage forms. Particularlyuseful is the administration of a compound of Formula I directly inparenteral formulations. The pharmaceutical compositions are prepared byconventional techniques appropriate to the desired preparationcontaining appropriate amounts of the active ingredient, that is, thecompound of Formula I according to the invention. See, for example,Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton,Pa., 17th edition, 1985.

The dosage of the compounds of Formula I to achieve a therapeutic effectwill depend not only on such factors as the age, weight and sex of thepatient and mode of administration, but also on the degree of potassiumchannel activating activity desired and the potency of the particularcompound being utilized for the particular disorder of diseaseconcerned. It is also contemplated that the treatment and dosage of theparticular compound may be administered in unit dosage form and that theunit dosage form would be adjusted accordingly by one skilled in the artto reflect the relative level of activity. The decision as to theparticular dosage to be employed (and the number of times to beadministered per day) is within the discretion of the physician, and maybe varied by titration of the dosage to the particular circumstances ofthis invention to produce the desired therapeutic effect.

A suitable dose of a compound of Formula I or pharmaceutical compositionthereof for a mammal, including man, suffering from, or likely to sufferfrom any condition as described herein is an amount of active ingredientfrom about 0.1 μg/kg to 100 mg/kg body weight. For parenteraladministration, the dose may be in the range of 1 μg/kg to 10 mg/kg bodyweight for intravenous administration The active ingredient willpreferably be administered in equal doses from one to four times a day.However, usually a small dosage is administered, and the dosage isgradually increased until the optimal dosage for the host undertreatment is determined. A suitable dose for the treatment of infectiousdisease in humans will preferably range from about 100 mg to about 1,000mg of the active ingredient for a 70 kg adult, depending on the natureof the infection and the frequency and route of administration interalia.

However, it will be understood that the amount of the compound actuallyadministered will be determined by a physician, in the light of therelevant circumstances including the condition to be treated, the choiceof compound of be administered, the chosen route of administration, theage, weight, and response of the individual patient, and the severity ofthe patient's symptoms.

The following examples are given by way of illustration and are not tobe construed as limiting the invention in any way inasmuch as manyvariations of the invention are possible within the spirit of theinvention.

DESCRIPTION OF SPECIFIC EMBODIMENTS

In the following examples, all temperatures are given in degreesCentigrade. Melting points were recorded on a Gallenkamp capillarymelting point apparatus are uncorrected. Proton magnetic resonance (¹ HNMR) and carbon magnetic resonance (¹³ C NMR) spectra were recorded on aBruker AC 300 spectrometer. All spectra were determined in the solventsindicated and chemical shifts are reported in 6 units downfield from theinternal standard tetramethylsilane (TMS) and interproton couplingconstants are reported in Hertz (Hz). Splitting patterns are designatedas follows: s, singlet; d, doublet; t, triplet; q, quartet; m,multiplet; br, broad peak; dd, doublet of doublet; bd, broad doublet;dt, doublet of triplet; bs, broad singlet; dq, doublet of quartet.Infrared (IR) spectra using potassium bromide (KBr) were determined on aPerkin Elmer 781 spectrometer from 4000 cm⁻¹ to 400 cm⁻¹, calibrated to1601 cm⁻¹ absorption of a polystyrene film and reported in reciprocalcentimeters (cm⁻¹). Ultraviolet spectra were determined by RobertsonMicrolit Laboratories, Inc. in the solvents indicated. Low resolutionmass spectra (MS) and the apparent molecular (MH⁺) was determined on aFinnigan TSQ 7000. The element analysis are reported as percent byweight.

The following procedures Nos. 1-3 illustrate representative proceduresfor the preparation of intermediates and methods for the preparation ofproducts according to this invention. It should also be evident to thoseskilled in the art that appropriate substitution of both the materialsand methods disclosed herein will produce the examples illustrated belowand those encompassed by the scope of this invention.

PROCEDURE 1

General Method for Compounds of Formula III wherein

    R.sup.a =CH.sub.2 OCH.sub.3

Sodium hydride (850 mg, 60% in mineral oil, 22 mmoles) was rinsed withhexanes (2×5 mL), then DMF (25 mL) was added and the temperature of thesuspension was kept at 0°-5° C. Isatin reagent of formula III whereinR^(a) =H (20 mmoles) was added into the NaH/DMF suspension in smallportions and the resulting dark solution was stirred for 20 minutes.Bromomethyl methyl ether (22 mmoles, 1.1 eq.) was added via a plasticsyringe in one portion and the reaction mixture was allowed to stir atroom temperature for 16 hours. The crude mixture was poured into water(250 mL), and precipitated product was collected by filtration, washedand dried to give a compound of formula III wherein R^(a) =CH₂ OCH₃.

PROCEDURE 2

General Method for Preparation of Compounds of Formula Ia

    (R.sup.a =H, CH.sub.3 or --CH.sub.2 OCH.sub.3)

A mixture of isatin of formula III (30 mmol), the compound of formula IV(30 mmol) and NaOMe (90 mL of 1.0M solution in MeOH, 3 equiv.) in MeOH(150 mL) was heated at reflux for 3-12 hours. When TLC indicatedcomplete conversion, the reaction mixture was cooled to room temperatureand added to a 0.5N HCl solution (500 mL) with stirring. The productwhich precipitated out from the acidified mixture was collected byfiltration, washed with water (3×50 mL) and dried under reduced pressureto give a mixture of compounds of formula Ia and II. The solid mixturewas suspended in AcOEt (100 mL) and heated a reflux for about 20minutes. After cooling to room temperature, the mixture was filtered toremove the undesirable product of formula II. The filtrate wasevaporated to dryness and the residue was suspended and stirred in amixed solvent (100 mL) of AcOEt and hexane (1:4) for about 10 minutes,filtered and dried to produce the compounds of formula Ia. In somemixtures when the ratio of the desired compound of formula Ia is high,filtration of the AcOEt suspension will yield the desired product offormula Ia. In other cases, the compound of formula Ia can be obtainedby flash column chromatography (silica gel, AcOEt/Hexane: 10-30%) of themixture.

PROCEDURE 3

General Method for Preparation of Compounds of Formula Ib

To a suspension of 6 mmol of methyl ether of formula Ia in methylenechloride was added 30 mL of a solution of BBr₃ (1.0M in CH₂ Cl₂, 5.0equiv.) at -78° C. where upon the reaction became homogenous. Thesolution was stirred at -78° C. for about 3 hours, the bath was removedand stirring was continued for an additional 20 hours at roomtemperature. Water (0.5 mL) was added and the reaction was stirred for10 minutes. The solvents were removed in vacuo to give a solid residuewhich was suspended in 100 mL of water, sonicated for 5-10 minutes, thenstirred for 10 minutes, filtered, washed with water (3×30 mL) and driedto produce a compound of formula Ib in nearly quantitative yield.

EXAMPLE 1

5,7-Dichloro-4-(5-chloro-2-methoxyphenyl)-3-hydroxy-2(1H)-quinolinoneand its regioisomer5,7-dichloro-3-(5-chloro-2-methoxyphenyl)-4-hydroxy-2(1H)-guinolinone

A mixture of 4,6-dichloro-1H-indol-2,3-dione (6.48 g, 0.03 mol),5-chloro-2-methoxy- N-(4-methylphenyl)hydrazonomethyl!phenyl (10.65 g,0.0315 mol, 1.05 eq.) and NaOMe (90 mL of 1.0M solution in methanol) inmethanol (150 mL) was heated at reflux temperature for 3 hours. Thereaction mixture was cooled and the solid was collected by filtrationand washed with methanol (3×10 mL). The solid was suspended in 0.5N HClsolution (500 mL), stirred for 20 minutes then filtered, washed withwater (3×50 mL) and dried to yield 2.76 g of the desired isomer5,7-dichloro-4-(5-chloro-2-methoxyphenyl)-3-hydroxy-2(1H)-quinolinone.

¹ H NMR (300 MHz, DMSO-d₆) δ: 7.39 (dd,1H, J=2.7, 8.8 Hz), 7.36, (1H, d,J=2.2 Hz), 7.23 (1H, d, J=2.2 Hz), 7.15 (1H, d, J=2.2 Hz), 7.02 (1H, d,J=8.9 Hz), 3.64 (3H, s), 12.53 (1H, bs), 9.79 (1H, bs); ¹³ C NMR (75MHz, DMSO-d₆) δ: 157.2, 156.0, 145.4, 135.4, 130.2, 130.0, 130.0, 128.8,126.3, 124.5, 123.6, 118.5, 116.6, 114.4, 112.2, 55.7; UV(abs. ethanolat 5.2×10⁻⁴ g/100 mL) λ_(max) : 232 (1107), 336 (299), 288 (292), 322(289) and 310 (221); MS (DCl): 370 (MH⁺); IR (KBr, cm⁻¹): 3500-2400,1665, 1300-1200 and 1020; Anal. calcd. for C₁₆ H₁₀ Cl₃ NO₃ : C, 51.85;H, 2.72; N, 3.78; Found: C, 51.89; H, 2.81; N, 3.74.

The filtrate from the above reaction mixture was added to 0.5N HClsolution (1500 mL) with stirring. The product which precipitated fromthe acidified mixture was collected by filtration and dried to yield8.11 g of a mixture (6:1) of the regioisomer5,7-dichloro-3-(5-chloro-2-methoxyphenyl)-4-hydroxy-2(1H-quinolinone andsome of the desired product. A sample of the purified regioisomer hadthe following characteristics:

¹ H NMR (300 MHz, DMSO-d₆) δ: 11.70 (1H, s), 10.08 (1H, s), 7.37 (1H,dd, J=2.7, 8.9 Hz), 7.29 (2H, d, J=1.6 Hz), 7.13 (1H, d, J=2.6 Hz), 7.05(1H, d, J=8.9 Hz), 3.68 (3H, s); ¹³ C NMR (75 MHz, DMSO-d₆) δ: 156.9,141.2, 134.3, 132.2, 131.6, 128.9, 124.1, 123.6, 113.8, 113.1, 55.7;UV(abs. ethanol at 4.8×10⁻⁴ g/100 mL) λ_(max) : 234 (1480), 296 (373)and 326 (300) nm; MS (DCl): 370 (MH⁺); IR (KBr, cm⁻¹): 3500-2500, 1660and 1250; Anal. calcd. for C₁₆ H₁₀ Cl₃ NO₃ : C, 51.85; H, 2.72; N, 3.78;Found: C, 52.01; H, 2.76; N, 3.80.

EXAMPLE 2

5,7-Dichloro-4-(5-chloro-2-hydroxyphenyl)-3-hydroxy-2(1H)-quinolinone

To a suspension of5,7-dichloro-4-(5-chloro-2-methoxyphenyl)-3-hydroxy-2(1H)-quinolinone(2.22 g, 6.0 mmol) prepared in Example 1! in methylene chloride wasadded BBr₃ (30 mL, 1.0M solution in methylene chloride, 5.0 equiv.) at-78° C. and the suspension became a clear solution. The solution wasstirred under an argon atmosphere at -78° C. for 3 hours then at roomtemperature for an additional 20 hours. Distilled water (0.5 mL) wasadded dropwise and stirring continued for 10 minutes. The reactionmixture was evaporated under vacuum and the solid residue was suspendedin water (100 mL), sonicated for 5 minutes then stirred for 10 minutes,filtered, washed with water (3×30 mL) and dried to yield 2.15 g (≈100%)of the title compound as a white solid: mp=297°-299° C.;

Anal. calcd. for C₁₅ H₈ NO₃ Cl₃ : C, 48.56; H, 2.61; N, 3.78; Found: C,48.64; H, 2.52; N, 3.74; IR (KBr, cm⁻¹): 3600-2000, 1660 and 1250;UV(abs. ethanol at 5.2×10⁻⁴ g/100 mL) λ_(max) : 232 (1066), 336 (314),322 (304), 290 (299) and 684 (3.5); ¹ H NMR (300 MHz, DMSO-d₆) δ: 12.50,9.62, 7.35 (1H, d, J=2.2 Hz), 7.22 (1H, d, J=2.4 Hz), 7.19 (1H, dd,J=2.7; 8.7 Hz), 7.05 (1H, d, J=2.7 Hz), 6.81 (1H, d, J=8.7 Hz); ¹³ C NMR(75 MHz, DMSO-d₆) δ: 157.39, 154.14, 145.53, 135.47, 130.17, 129.78,128.50, 124.55, 124.79, 121.79, 118.74, 116.87, 116.32, 114.25.

A sample of the title compound was crystallized from EtOAc/H₂ O to yieldcolorless rod crystals. The structure and solid state conformationassigned to the title compound was confirmed by single crystal x-rayanalysis. The dihedral angle between the plane of the quinoline and theplane of the phenyl substitutent is 100.23(6)°.

EXAMPLES 3-18

Following the general procedures Nos. 2 and 3, and the representativeExamples 1 and 2, the following 4-aryl-3-hydroxyquinolin-2-one productsare made using the appropriate intermediates of formulas III and IV toproduce the compounds of formula Ia and Ib as illustrated for Examples 3to 18 in Table II.

In general, the H¹ NMR spectra in DMSO-d₆ of the desired productexhibited a chemical shift for the 3-OH peak at about 9.5-9.8 δ and forthe NH peak at about 12.2-12.6 δ.

                                      TABLE II    __________________________________________________________________________     ##STR7##    Example                Melting                                Yield                                   Calculated (%)                                           Found (%)    No.  R  R.sup.1              R.sup.2                 R.sup.3                    R.sup.4                      R.sup.5                         R.sup.6                           Pt.  (%)                                   C; H; N C; H; N    __________________________________________________________________________    3    H  H NO.sub.2                 H  H CH.sub.3                         Br                           >330° C.                                92.1                                   49.13; 2.83; 7.16                                           48.97; 2.64; 7.07    4    H  Cl              H  Cl H CH.sub.3                         Br                           306-307                                92.2                                   46.30; 2.43; 3.37                                           46.34; 2.36; 3.34    5    H  H H  Cl H CH.sub.3                         Cl                           303-305                                97.5                                   57.17; 3.30; 4.17                                           56.90; 3.35; 3.93    6    H  H H  H  H H  Br                           220-221                                95.8                                   54.24; 3.03; 4.22                                           53.85; 3.11; 4.11    7    CH.sub.3            H H  H  H H  Br                           301-302                                83.5                                   55.51; 3.49; 4.05                                           54.93; 3.37; 3.88    8    H  H H  H  H H  Cl                           336-338                                77.3                                   61.46; 3.65; 4.78                                           61.57; 3.48; 4.62    9    H  H NO.sub.2                 H  H H  Br                           >330° C.                                96.0                                   47.09; 2.53; 7.32                                           47.09; 2.15; 7.02    10   H  H H  CF.sub.3                    H H  Cl                           224-225                                92.3                                   52.17; 2.85; 3.80                                           52.07; 2.85; 3.77    11   H  H H  CF.sub.3                    H H  Br                           182 dec                                92.8                                   46.55; 2.54; 3.29                                           46.56; 2.48; 3.31    12   H  Cl              H  Cl H H  Br                           292 dec                                95.6                                   43.93; 2.21; 3.42                                           43.97; 2.08; 3.30    13   H  H Cl H  H H  Cl                           >335° C.                                93.0                                   54.70; 3.00; 4.25                                           54.63; 2.73; 4.10    14   H  Cl              H  H  H H  Cl                           295-297                                90.4                                   55.31; 2.91; 4.30                                           55.27; 2.66; 4.19    15   H  H H  Cl H H  Cl                           281-283                                93.1                                   54.11; 3.09; 4.21                                           53.90; 2.72; 4.13    16   H  H CF.sub.3                 H  H H  Cl                           313-315                                66.9                                   54.03; 2.55; 3.94                                           53.86; 2.24; 3.72    17   H  H CF.sub.3                 H  H H  Br                           308-310                                84.7                                   48.03; 2.27; 3.50                                           47.61; 2.52; 3.29    18   CH.sub.3            Cl              H  Cl H H  Cl                           265-267                                82.6                                   51.85; 2.72; 3.78                                           51.69; 2.63; 3.43    __________________________________________________________________________

EXAMPLES 19-21

Following the general procedures Nos. 1, 2 and 3, wherein R^(a) is CH₂OCH₃, and the representative Examples 1 and 2, the following4-aryl-3-hydroxyquinolin-2-one products are made using the appropriateintermediates of formulas III and IV to produce the compounds of formulaIb as illustrated for Examples 19 to 21 in Table III.

                                      TABLE III    __________________________________________________________________________     ##STR8##    Example                Melting                                Yield                                   Calculated (%)                                           Found (%)    No.  R  R.sup.1              R.sup.2                 R.sup.3                    R.sup.4                      R.sup.5                         R.sup.6                           Pt.  (%)                                   C; H; N C; H; N    __________________________________________________________________________    19   H  H Br H  H H  Br                           >330° C.                                93.1                                   43.08; 2.36; 3.35                                           43.08; 2.03; 3.28    20   H  H Br H  H H  Cl                           >330° C.                                75.4                                   49.15; 2.47; 3.82                                           49.08; 2.56; 3.64    21   H  H H  H  Cl                      H  Cl                           >286-288                                90.1                                   54.41; 3.04; 4.23                                           54.54; 2.96; 4.02    __________________________________________________________________________

What is claimed is:
 1. A compound of the formula ##STR9## wherein R ishydrogen or methyl;R¹, R², R³ and R⁴ each are independently hydrogen,bromo, chloro or trifluoromethyl, and when R¹, R³ and R⁴ are hydrogen,R² is nitro; with the proviso that R¹, R², R³ and R⁴ are not allhydrogen; R⁵ is hydrogen or methyl; and R⁶ is bromo or chloro;or anontoxic pharmaceutically acceptable salt thereof.
 2. A compound ofclaim 1 wherein R is hydrogen and R⁵ is hydrogen; or a nontoxicpharmaceutically acceptable salt thereof.
 3. A compound of claim 1wherein R² is bromo, chloro, trifluoromethyl or nitro and R⁶ is bromo orchloro; or a nontoxic pharmaceutically acceptable salt thereof.
 4. Acompound of claim 1 wherein R¹ and R³ each are independently chloro andR⁶ is bromo or chloro; or a nontoxic pharmaceutically acceptable saltthereof.
 5. A compound of claim 1 selected from the group consistingof:5,7-dichloro-4-(5-chloro-2-hydroxyphenyl)-3-hydroxy-2(1H)-quinolinone;6-nitro-4-(5-bromo-2-hydroxyphenyl)-3-hydroxy-2(1H)-quinolinone;7-trifluoromethy-4-(5bromo-2-hydroxyphenyl)-3-hydroxy-2(1H)-quinolinone;5,7-dichloro-4-(5-bromo-2-hydroxyphenyl)-3-hydroxy-2(1H)-quinolinone;6-bromo-4-(5-bromo-2-hydroxyphenyl)-3-hydroxy-2(1H)-quinolinone;6-bromo-4-(5-chloro-2-hydroxyphenyl)-3-hydroxy-2(1H)-quinolinone;6-trifluoromethyl-4-(5-chloro-2-hydroxyphenyl)-3-hydroxy-2(1H)-quinolinone;and6-trifluoromethyl-4-(5-bromo-2-hydroxyphenyl)-3-hydroxy-2(1H)-quinolinone.6. A compound of claim 1 selected from the group consistingof:5,7-dichloro-4-(5-bromo-2-methoxyphenyl)-3-hydroxy-2(1H)-quinolinone;4-(5-bromo-2-hydroxyphenyl)-3-hydroxy-1-methylquinolin-2-one;7-trifluoromethyl-4-(5-chloro-2-hydroxyphenyl)-3-hydroxy-2(1H)-quinolinone;7-chloro-4-(5-chloro-2-hydroxyphenyl)-3-hydroxy-2(1H)-quinolinone; and5,7-dichloro-4-(5-chloro-2-hydroxyphenyl)-3-hydroxy-1-methylquinolin-2-one.7. A pharmaceutical composition for the treatment of disordersresponsive to openers of the large conductance calcium-activatedpotassium channels comprising a therapeutically effective amount of thecompound of claim 1 in association with a pharmaceutically acceptablecarrier or diluent.
 8. A pharmaceutical composition for the treatment ofdisorders responsive to openers of the large conductancecalcium-activated potassium channels comprising a therapeuticallyeffective amount of the compound of claim 5 in association with apharmaceutically acceptable carrier or diluent.